Reduced-viscosity concentrated protein formulations

ABSTRACT

The present application concerns concentrated protein formulations with reduced viscosity, which arc particularly suitable for subcutaneous administration. The application further concerns a method for reducing the viscosity of concentrated protein formulations.

RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 11/068,553, filedFeb. 28, 2005, now pending, which is a divisional of Ser. No.09/971,511, filed Oct. 4, 2001, now U.S. Pat. No. 6,875,432, whichclaims the benefit under 35 U.S.C. §119 of both U.S. Ser. No.60/293,834, filed May 24, 2001, and U.S. Ser. No. 60/240,107, filed Oct.12, 2000; the contents all of which are incorporated herein byreference.

BACKGROUND OF THE INVENTION

Field of the Invention

This invention pertains to concentrated protein formulations withreduced viscosity, which are particularly suitable for subcutaneousadministration. The invention further concerns a method of reducingviscosity of concentrated protein formulations.

Description of the Related Art

In the past ten years, advances in biotechnology have made it possibleto produce a variety of proteins for pharmaceutical applications usingrecombinant DNA techniques. Because proteins are larger and more complexthan traditional organic and inorganic drugs (i.e. possessing multiplefunctional groups in addition to complex three-dimensional structures),the formulation of such proteins poses special problems. One of theproblems is the elevated viscosity values of protein formulations,especially at high concentration. The delivery of high proteinconcentration is often required for subcutaneous administration due tothe volume limitations 1.5 ml) and dose requirements (usually 50 mg,preferably 100 mg). For example, if a protein is to be administered topatients at 2 mg/kg on a weekly basis, the average weekly dose will be130 mg considering 65 kg as the average weight of patients. Sinceinjection volumes of more than 1.5 ml are not well tolerated forsubcutaneous administration, the protein concentration for a weeklysubcutaneous administration would have to be approximately 100 mg/ml(130 mg protein in less than 1.5 ml volume). However, highlyconcentrated protein formulations pose several problems. One problem isthe tendency of proteins to form particulates during processing and/orstorage, which makes manipulation during further processing difficult.In the case of reconstituted liquid formulations, this is usuallycircumvented by adding a suitable surfactant (e.g. a polysorbate) duringlyophilization or after lyophilization while reconstituting theformulation. Although surfactants have been shown to significantlyreduce the degree of particulate formation of proteins, they do notaddress another problem associated with manipulating and administeringconcentrated protein formulations. Proteins tend to form viscoussolutions at high concentration because of their macromolecular natureand potential for intermolecular interactions. Moreover, many proteinsare often lyophilized in the presence of large amounts oflyoprotectants, such as sugar to maintain their stability. The sugar canenhance the intermolecular interactions and increase the viscosity.Highly viscous formulations are difficult to manufacture, draw into asyringe and inject subcutaneously. The use of force in manipulating theviscous formulations leads to excessive frothing, and the resultantdetergent-like action of froth has the potential to denature andinactivate the therapeutically active protein. Moreover, viscoussolution increases the back-pressure during UF/DF process and makesrecovery of protein difficult. This can result in considerable loss ofprotein product. Satisfactory solution of this problem is lacking in theprior art. Therefore, there is a need to develop a method of reducingthe viscosity of a formulation containing high concentration of protein.

Stable isotonic lyophilized protein formulations are disclosed in PCTpublication WO 97/04801, published on Feb. 13, 1997, the entiredisclosure of which is hereby expressly incorporated by reference. Thedisclosed lyophilized formulations can be reconstituted to generate highprotein-concentration liquid formulations without apparent loss ofstability. However, the potential issues associated with the highviscosity of the reconstituted formulations are not addressed.

Applicants have discovered that the preparation of proteinaceous,lyophilized formulation with 100 mM NaCl diluent can result in aslightly hypertonic solution. It had been previously believed thatpharmaceutical formulations must be maintained at physiological pH andbe isotonic. This belief was based at least in part on the perceptionthat the administration of hypertonic formulation could lead todehydration and therefore could damage the tissue at the site ofinjection. However, the belief of the requirement for absoluteisotonicity of a pharmaceutical formulation may not be well-founded. Forexample, Zietkiewicz et al., Grzyby Drozdzopodobne 23: 869-870 (1971)have shown that absolute isotonicity of the drugs is not necessary. Itwas found to be sufficient to avoid the drug solutions that exceed thecritical limits of hypertonicity. For example, tissue damage wasobserved only when hypertonic solution of 1300 mOsmol/Kg (˜650 mM NaCl)or higher was administered subcutaneously or intramuscularly toexperimental animals. As a result, formulations which arc slightlyhypertonic, or outside of the physiological pH range do not appear topresent a risk of tissue damage at the site of administration.

Applicants have further found that proteinaceous solutions having alowered (4.0-5.3) or elevated (6.5-12.0) pH was also effective atreducing the viscosity of high concentration protein formulations.

The present invention is directed to providing a high concentrationprotein formulation with reduced viscosity, which is easy to handle andis suitable for subcutaneous administration. The present invention isfurther directed to providing a method of reducing viscosity ofconcentrated protein formulations.

SUMMARY OF THE INVENTION

The present invention concerns a method of lowering the viscosity ofconcentrated protein composition by: (1) increasing the total ionicstrength of the formulation through the addition of salts or buffercomponents; or (2) altering the pH of the formulation to be lower (≈4.0to ≈5.3) or elevated ≈6.5 to ≈12.0), without significantly compromisingstability or biological activity. Accordingly, the invention concernsmethods and means for reducing the viscosity of concentrated proteinformulations, primarily to ensure easy manipulation before and duringadministration to a patient.

In one aspect, the present invention provides a stable formulation ofreduced viscosity comprising a protein in an amount of at least about 80mg/ml and a salt or a buffer in an amount of at least about 50 mM, andhaving a kinematic viscosity of about 50 cs or less. The salts and/orbuffers are pharmaceutically acceptable and are derived from variousknown acids (inorganic and organic) or base forming metals and amines.Alternatively, the salts and/or buffers may be derived from amino acids.In a specific aspect, the salts are chosen from the group consisting ofsodium chloride, arginine hydrochloride, sodium thiocyanate, ammoniumthiocyanate, ammonium sulfate, ammonium chloride, calcium chloride, zincchloride and sodium acetate. In another aspect, the salts or buffers aremonovalent. In yet another aspect, the formulation contains the abovesalt or buffer components in an amount of about 50-200 mM, and has aviscosity of about 2 to 30 cs. In a particular embodiment, the proteinin the formulation has a molecular weight of at least about 15-20 kD. Inanother particular embodiment, the formulation is hypertonic. In yetanother particular aspect, the formulation may further comprise asurfactant such as polysorbatc. The invention also contemplates areconstituted formulation that further comprises a lyoprotectant such assugars. In yet another particular aspect, the lyoprotectant sugar canbe, for example, sucrose or trehalose, and may be present in an amountof about 60-300 mM. In another specific aspect, the proteinconcentration in the reconstituted formulation is about 2-40 timesgreater than the protein concentration in the mixture beforelyophilization.

In another embodiment, the invention provides a stable formulation ofreduced viscosity comprising a protein in an amount of at least about 80mg/ml by having a pH lower (≈4.0 to ≈5.3) or elevated (≈6.5 to ≈12.0),wherein the kinematic viscosity is reduced to 50 cs or less. In aspecific aspect, the viscosity is reduced to about 2 to 30 cs. Inanother specific aspect, the pH is altered through the addition of apharmaceutically acceptable acid, base or buffer, and is added in anamount of at least about 10 mM, preferably about 50-200 mM, morepreferably about 100-200 mM, most preferably about 150 mM. In a specificaspect, the acid, base and/or buffers are monovalent. In anotherspecific aspect, the acid, base and/or buffers are selected from thegroup consisting of acetic acid, hydrochloric acid, and arginine. Inanother particular aspect, the formulation may further comprise asurfactant such as polysorbatc. The invention also contemplates areconstituted formulation that further comprises a lyoprotectant such assugars. In a particular aspect, the lyoprotectant sugar can be, forexample, sucrose or trehalose, and may be present in an amount of about60-300 mM. In another preferred aspect, the protein concentration in thereconstituted formulation is about 2-40 times greater than the proteinconcentration in the mixture before lyophilization. In a particularaspect, the pH is any tenth pH value within those enumerated above; forexample, for the lower pH value, example values are pH 4.0, 4.1, 4.2,4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2 and 5.3. At the higherpH range, example values are 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2,7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6,8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0,10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11.0, 11.1, 11.2,11.3, 11.4, 11.5, 11.6, 11.7, 11.8, 11.9 and 12.0.

In a particular embodiment, the invention provides a formulationcontaining high concentrations of large molecular weight proteins, suchas immunoglobulins. The immunoglobulins may, for example, be antibodiesdirected against a particular predetermined antigen. In a specificaspect, the antigen is IgE (e.g., rhuMAbE-25, rhuMAbE-26 and rhuMAbE-27described in WO 99/01556). Alternatively, the antigen may include: theCD proteins CD3, CD4, CD8, CD19, CD20 and CD34; members of the HERreceptor family such as EGF receptor, HER2, HER3 or HER4 receptor; celladhesion molecules such as LFA-1, Mol, p150,95, VLA-4, ICAM-1, VCAM andαv/β3 integrin including the α- and β-subunits thereof (e.g.,anti-CD11a, anti-CD18 or anti-CD11b antibodies); growth factors such asVEGF; blood group antigens; flk2/flt3 receptor; obesity (OB) receptor;and protein C.

The formulations of the present invention may be pharmaceuticalformulations, in particular, formulations for subcutaneousadministration.

In another aspect, the invention provides a method of reducing theviscosity of a formulation containing a protein in an amount of at leastabout 80 mg/ml by the addition of a salt or buffer component in anamount of at least about 50 mM, wherein the kinematic viscosity isreduced to 50 cs or less. In a specific aspect, the viscosity is reducedto about 2 to 30 cs. In another specific aspect, the salts or buffercomponents may be added in an amount of at least about 100 mM,preferably about 50-200 mM, more preferably about 100-200 mM, mostpreferably about 150 mM. The salts and/or buffers are pharmaceuticallyacceptable and are derived from various known acids (inorganic andorganic) with “base forming” metals or amines. Alternatively, the saltsand/or buffers may be derived from amino acids. In yet another specificaspect, the salts and/or buffers are monovalent. In yet another specificaspect, the salts are selected from the group consisting of sodiumchloride, arginine hydrochloride, sodium thiocyanate, ammoniumthiocyanate, ammonium sulfate, ammonium chloride, calcium chloride, zincchloride and sodium acetate. In yet another aspect, the formulationcontains the above salt or buffer components in an amount of about50-200 mM, and has a viscosity of about 2 to 30 cs. In yet anotheraspect, the protein in the formulation has a molecular weight of atleast about 15-20 kD. In another particular embodiment, the formulationmay further comprise a surfactant such as polysorbate. The inventionalso contemplates a reconstituted formulation that further comprises alyoprotectant such a sugar. In a particular aspect, the lyoprotectantsugar can be, for example, sucrose or trehalose, and may be present inan amount of about 60-300 mM. In a specific aspect, the formulation canbe reconstituted with a diluent comprising the buffer or salt. In apreferred embodiment, the protein concentration in the reconstitutedformulation is about 2-40 times greater than the protein concentrationin the mixture before lyophilization. In yet another embodiment, theinvention provides a method for reducing the viscosity comprising aprotein in an amount of at least about 80 mg/ml by altering the pH to belower (≈4.0 to ≈5.3) or elevated to ≈6.5 to ≈12.0), wherein thekinematic viscosity is reduced to 50 cs or less. In a specific aspect,the viscosity is reduced to about 2 to 30 cs. In another specificaspect, the pH is altered through the addition of a pharmaceuticallyacceptable acid, base or buffer, and is added in an amount of at leastabout 10 mM, preferably about 50-200 mM, more preferably about 100-200mM, most preferably about 150 mM. In a specific aspect, the acid, baseand/or buffers are monovalent. In an another specific aspect, the acid,base and/or buffers are selected from the group consisting of aceticacid, hydrochloric acid, and arginine In another particular embodiment,the formulation may further comprise a surfactant such as polysorbate.The invention also contemplates a reconstituted formulation that furthercomprises a lyoprotectant such as sugars. In a particular aspect, thelyoprotectant sugar can be, for example, sucrose or trehalose, and maybe present in an amount of about 60-300 mM. In a preferred embodiment,the protein concentration in the reconstituted formulation is about 2-40times greater than the protein concentration in the mixture beforelyophilization. In a particular aspect, the pH is any tenth pH valuewithin those enumerated above; for example, for the lower pH value,example values are pH 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9,5.0, 5.1, 5.2 and 5.3. At the higher pH range, example values are 6.5,6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9,8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3,9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6,10.7, 10.8, 10.9, 11.0, 11.1, 11.2, 11.3, 11.4, 11.5, 11.6, 11.7, 11.8,11.9 and 12.0.

In yet another aspect, the invention provides a method of reducing theviscosity of a formulation of a protein having a molecular weight of atleast about 15-20 kD, including immunoglobulins, specifically antibodieswhich specifically bind to a particular antigen. In a specific aspect,the method is used to prepare a reconstitutable formulation, especiallythose that are concentrated to a much greater concentration oftherapeutic protein (e.g., 2-40 times) after the concentration step(e.g., lyophilization) compared to before.

In yet another embodiment, the invention provides a method for thetreatment, prophylactic or therapeutic, of a disorder treatable by theprotein (e.g. antibody) formulated, using the formulations disclosedherein. Such formulations are particularly useful for subcutaneousadministration. Also provided is an article of manufacture comprising acontainer enclosing a formulation disclosed herein.

In yet another embodiment, the present invention discloses a method ofpreventing self-association of proteins in concentrated liquidformulations by (1) adding a salt or a buffer component in an amount ofat least about 50 mM; or (2) altering the pH by lowering to (≈4.0 to≈5.3) or elevating to (≈6.5 to ≈12.0). In a specific aspect, theself-association to be prevented is that induced or exacerbated by thepresence of sugars (e.g., sucrose or trehalose) that are commonly usedas lyoprotectants. Accordingly, this method is particularly useful forpreventing self-association of reconstituted lyophilized formulations.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the effects of protein concentration on viscosity ofreconstituted formulation containing the anti-IgE antibody rhuMAb E25,16 mM histidine, 266 mM sucrose and 0.03% Polysorbate 20 at 25° C.

FIG. 2 depicts the effects of NaCl concentration on viscosity ofreconstituted formulation containing 125 mg/ml of the antibody IgEantibody rhuMAb E25, 16 mM histidine, 266 mM sucrose, 0.03% Polysorbate20 and various amounts of NaCl at 25° C.

FIG. 3 shows the effects of various salts on viscosity of reconstitutedformulation containing 40 mg/ml of the anti-IgE antibody rhuMAb E25, 10mM histidine, 250 mM sucrose, 0.01% Polysorbate 20 and various amountsof salts at 25° C.

FIG. 4 shows the effects of buffer concentration on viscosity of aliquid formulation containing 80 mg/ml of the anti-IgE antibody rhuMAbE25, 50 mM histidine, 150 mM trehalose, 0.05% Polysorbate 20 and variousamounts of histidine, acetate, or succinate components at 25° C.

FIG. 5 shows the effects of NaCl concentration on viscosity of areconstituted formulation containing 21 mg/ml rhuMAb E26, 5 mMhistidine, 275 mM sucrose at 6° C.

FIG. 6 shows the effects of pH on viscosity of liquid formulationscontaining 130 mg/ml rhuMAb E25, 2-17.5 mM of acetate or arginine withand without 150 mM NaCl at 25° C.

FIG. 7 shows the effects of pH on viscosity of reconstituted lyophilizedformulations containing 94 mg/ml rhuMAb E25, 250 mM trehalose, 20 mMhistidine, at 25° C.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

I. Definitions

By “protein” is meant a sequence of amino acids for which the chainlength is sufficient to produce the higher levels of tertiary and/orquaternary structure. Thus, proteins are distinguished from “peptides”which are also amino acid—based molecules that do not have suchstructure. Typically, a protein for use herein will have a molecularweight of at least about 15-20 kD, preferably at least about 20 kD.

Examples of proteins encompassed within the definition herein includemammalian proteins, such as, e.g., growth hormone, including humangrowth hormone and bovine growth hormone; growth hormone releasingfactor; parathyroid hormone; thyroid stimulating hormone; lipoproteins;α-1-antitrypsin; insulin A-chain; insulin B-chain; proinsulin; folliclestimulating hormone; calcitonin; luteinizing hormone; glucagon; clottingfactors such as factor VIIIC, factor IX, tissue factor, and von

Willebrands factor; anti-clotting factors such as Protein C; atrialnatriuretic factor; lung surfactant; a plasminogen activator, such asurokinase or tissue-type plasminogen activator (t-PA,e.g., Activase®,TNKase®, Retevase®); bombazine; thrombin; tumor necrosis factor-α and-β; enkephalinase; RANTES (regulated on activation normally T-cellexpressed and secreted); human macrophage inflammatory protein(MIP-1-α); serum albumin such as human scrum albumin;mullerian-inhibiting substance; relaxin A-chain; relaxin B-chain;prorelaxin; mouse gonadotropin-associated peptide; DNase; inhibin;activin; vascular endothelial growth factor (VEGF); receptors forhormones or growth factors; an integrin; protein A or D; rheumatoidfactors; a neurotrophic factor such as bone-derived neurotrophic factor(BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6), or anerve growth factor such as NGF-β; platelet-derived growth factor(PDGF); fibroblast growth factor such as aFGF and bFGF; epidermal growthfactor (EGF); transforming growth factor (TGF) such as TGF-a and TGF-β,including TGF-β1, TGF-β2, TGF-β3, TGF-β4, or TGF-β5; insulin-like growthfactor-I and -II (IGF-I and IGF-II); des(1-3)-IGF-I (brain IGF-I);insulin-like growth factor binding proteins; CD proteins such as CD3,CD4, CD8, CD19 and CD20; erythropoietin (EPO); thrombopoietin (TPO);osteoinductive factors; immunotoxins; a bone morphogenetic protein(BMP); an interferon such as interferon-α, -β, and -γ; colonystimulating factors (CSFs), e.g., M-CSF, GM-CSF, and G-CSF; interleukins(ILs), e.g., IL-1 to IL-10; superoxide dismutase; T-cell receptors;surface membrane proteins; decay accelerating factor (DAF); a viralantigen such as, for example, a portion of the AIDS envelope; transportproteins; homing receptors; addressins; regulatory proteins;immunoadhesins; antibodies; and biologically active fragments orvariants of any of the above-listed polypeptides.

The protein which is formulated is preferably essentially pure anddesirably essentially homogeneous (i.e. free from contaminatingproteins). “Essentially pure” protein means a composition comprising atleast about 90% by weight of the protein, based on total weight of thecomposition, preferably at least about 95% by weight.

“Essentially homogeneous” protein means a composition comprising atleast about 99% by weight of protein, based on total weight of thecomposition.

In certain embodiments, the protein is an antibody. The antibody maybind to any of the above-mentioned molecules, for example. Exemplarymolecular targets for antibodies encompassed by the present inventioninclude CD proteins such as CD3, CD4, CD8, CD19, CD20 and CD34; membersof the HER receptor family such as the EGF receptor, HER2, HER3 or HER4receptor; cell adhesion molecules such as LFA-1, Mol, p150,95, VLA-4,ICAM-1, VCAM and αv/β3 integrin including either α or β subunits thereof(e.g. anti-CD 11a, anti-CD18 or anti-CD1 lb antibodies); growth factorssuch as VEGF; IgE; blood group antigens; flk2/flt3 receptor; obesity(OB) receptor; protein C etc.

The term “antibody” is used in the broadest sense and specificallycovers monoclonal antibodies (including full length antibodies whichhave an immunoglobulin Fc region), antibody compositions withpolyepitopic specificity, bispecific antibodies, diabodies, andsingle-chain molecules, as well as antibody fragments (e.g., Fab,F(ab′)₂, and Fv).

The basic 4-chain antibody unit is a heterotetrameric glycoproteincomposed of two identical light (L) chains and two identical heavy (H)chains. An IgM antibody consists of 5 of the basic heterotetramer unitalong with an additional polypeptide called a J chain, and contains 10antigen binding sites, while IgA antibodies comprise from 2-5 of thebasic 4-chain units which can polymerize to form polyvalent assemblagesin combination with the J chain. In the case of IgGs, the 4-chain unitis generally about 150,000 daltons. Each L chain is linked to an H chainby one covalent disulfide bond, while the two H chains are linked toeach other by one or more disulfide bonds depending on the H chainisotype. Each H and L chain also has regularly spaced intrachaindisulfide bridges. Each H chain has at the N-terminus, a variable domain(V_(H)) followed by three constant domains (C_(H)) for each of the a andy chains and four C_(H) domains for g and c isotypes. Each L chain hasat the N-terminus, a variable domain (V_(L)) followed by a constantdomain at its other end. The V_(L) is aligned with the V_(H) and theC_(L) is aligned with the first constant domain of the heavy chain(C_(H)1). Particular amino acid residues arc believed to form aninterface between the light chain and heavy chain variable domains. Thepairing of a V_(H) and V_(L) together forms a single antigen-bindingsite. For the structure and properties of the different classes ofantibodies, see e.g., Basic and Clinical Immunology, 8th Edition, DanielP. Sties, Abba I. Terr and Tristram G. Parsolw (eds), Appleton & Lange,Norwalk, Conn., 1994, page 71 and Chapter 6.

The L chain from any vertebrate species can be assigned to one of twoclearly distinct types, called kappa and lambda, based on the amino acidsequences of their constant domains. Depending on the amino acidsequence of the constant domain of their heavy chains (CH),immunoglobulins can be assigned to different classes or isotypes. Thereare five classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, havingheavy chains designated α, δ, ε, γ and μ, respectively. The γ and μclasses are further divided into subclasses on the basis of relativelyminor differences in the CH sequence and function, e.g., humans expressthe following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2.

The term “variable” refers to the fact that certain segments of thevariable domains differ extensively in sequence among antibodies. The Vdomain mediates antigen binding and defines the specificity of aparticular antibody for its particular antigen. However, the variabilityis not evenly distributed across the entire span of the variabledomains. Instead, the V regions consist of relatively invariantstretches called framework regions (FRs) of about 15-30 amino acidresidues separated by shorter regions of extreme variability called“hypervariable regions” or sometimes “complementarity determiningregions” (CDRs) that are each approximately 9-12 amino acid residues inlength. The variable domains of native heavy and light chains eachcomprise four FRs, largely adopting a β-sheet configuration, connectedby three hypervariable regions, which form loops connecting, and in somecases forming part of, the β-sheet structure. The hypervariable regionsin each chain are held together in close proximity by the FRs and, withthe hypervariable regions from the other chain, contribute to theformation of the antigen binding site of antibodies (see Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md. (1991). Theconstant domains are not involved directly in binding an antibody to anantigen, but exhibit various effector functions, such as participationof the antibody dependent cellular cytotoxicity (ADCC).

The term “hypervariable region” (also known as “complementaritydetermining regions” or CDRs) when used herein refers to the amino acidresidues of an antibody which are (usually three or four short regionsof exteme sequence variability) within the V-region domain of animmunoglobulin which form the antigen-binding site and are the maindeterminants of antigen specificity. There are at least two methods foridentifying the CDR residues: (1) An approach based on cross-speciessequence variability (i.e., Kabat et al., Sequences of Proteins ofImmunological Interest (National Institute of Health, Bethesda, M S1991); and (2) An approach based on crystallographic studies ofantigen-antibody complexes (Chothia, C. et al., J. Mol. Biol. 196:901-917 (1987)). However, to the extent that two residue identificationtechniques define regions of overlapping, but not identical regions,they can be combined to define a hybrid CDR.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast toconventional (polyclonal) antibody preparations which typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theantigen. In addition to their specificity, the monoclonal antibodies areadvantageous in that they are synthesized by the hybridoma culture,uncontaminated by other immunoglobulins. The modifier “monoclonal”indicates the character of the antibody as being obtained from asubstantially homogeneous population of antibodies, and is not to beconstrued as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies to be used in accordancewith the present invention may be made by the hybridoma method firstdescribed by Kohler et al., Nature, 256: 495 (1975), or may be made byrecombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The“monoclonal antibodies” may also be isolated from phage antibodylibraries using the techniques described in Clackson et al., Nature,352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991),for example.

The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is(are) identical with or homologous to corresponding sequencesin antibodies derived from another species or belonging to anotherantibody class or subclass, as well as fragments of such antibodies, solong as they exhibit the desired biological activity (U.S. Pat. No.4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855(1984)).

An “intact” antibody is one which comprises an antigen-binding site aswell as a CL and at least the heavy chain domains, C_(H)1, C_(H)2 andC_(H)3.

An “antibody fragment” comprises a portion of an intact antibody,preferably the antigen binding and/or the variable region of the intactantibody. Examples of antibody fragments include Fab, Fab′, F(ab′)₂ andFv fragments; diabodies; linear antibodies (see U.S. Pat. No. 5,641,870,Example 2; Zapata et al., Protein Eng. 8(10): 1057-1062 [1995]);single-chain antibody molecules and multispecific antibodies formed fromantibody fragments.

Papain digestion of antibodies produced two identical antigen-bindingfragments, called “Fab” fragments, and a residual “Fc” fragment, adesignation reflecting the ability to crystallize readily. The Fabfragment consists of an entire L chain along with the variable regiondomain of the H chain (V_(H)), and the first constant domain of oneheavy chain (C_(H)1). Each Fab fragment is monovalent with respect toantigen binding, i.e., it has a single antigen-binding site. Pepsintreatment of an antibody yields a single large F(ab′)₂ fragment whichroughly corresponds to two disulfide linked Fab fragments havingdifferent antigen-binding activity and is still capable of cross-linkingantigen. Fab′ fragments differ from Fab fragments by having a fewadditional residues at the carboxy terminus of the C_(H)1 domainincluding one or more cysteines from the antibody hinge region. Fab′-SHis the designation herein for Fab' in which the cysteine residue(s) ofthe constant domains bear a free thiol group. F(ab′)₂ antibody fragmentsoriginally were produced as pairs of Fab′ fragments which have hingecysteines between them. Other chemical couplings of antibody fragmentsare also known.

The Fc fragment comprises the carboxy-terminal portions of both H chainsheld together by disulfides. The effector functions of antibodies aredetermined by sequences in the Fc region, the region which is alsorecognized by Fc receptors (FcR) found on certain types of cells.

“Fv” is the minimum antibody fragment which contains a completeantigen-recognition and -binding site. This fragment consists of a dimerof one heavy- and one light-chain variable region domain in tight,non-covalent association. From the folding of these two domains emanatesix hypervarible loops (3 loops each from the H and L chain) thatcontribute the amino acid residues for antigen binding and conferantigen binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

“Single-chain Fv” also abbreviated as “sFv” or “scFv” are antibodyfragments that comprise the VH and VL antibody domains connected into asingle polypeptide chain. Preferably, the sFv polypeptide furthercomprises a polypeptide linker between the V_(H) and V_(L) domains whichenables the sFv to form the desired structure for antigen binding. For areview of the sFv, see Pluckthun in The Pharmacology of MonoclonalAntibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, NewYork, pp. 269-315 (1994).

The term “diabodies” refers to small antibody fragments prepared byconstructing sFv fragments (see preceding paragraph) with short linkers(about 5-10) residues) between the V_(H) and V_(L) domains such thatinter-chain but not intra-chain pairing of the V domains is achieved,thereby resulting in a bivalent fragment, i.e. , a fragment having twoantigen-binding sites. Bispecific diabodies are heterodimers of two“crossover” sFv fragments in which the V_(H) and V_(L) domains of thetwo antibodies are present on different polypeptide chains. Diabodiesare described in greater detail in, for example, EP 404,097; WO93/11161; Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448(1993).

An antibody that “specifically binds to” or is “specific for” aparticular polypeptide or an epitope on a particular polypeptide is onethat binds to that particular polypeptide or epitope on a particularpolypeptide without substantially binding to any other polypeptide orpolypeptide epitope.

“Humanized” forms of non-human (e.g., murine) antibodies are chimericimmunoglobulins, immunoglobulin chains or fragments thereof (such as Fv,Fab, Fab′, F(ab′)₂ or other antigen-binding subsequences of antibodies)of mostly human sequences, which contain minimal sequence derived fromnon-human immunoglobulin. For the most part, humanized antibodies arehuman immunoglobulins (recipient antibody) in which residues from ahypervariable region (also CDR) of the recipient are replaced byresidues from a hypervariable region of a non-human species (donorantibody) such as mouse, rat or rabbit having the desired specificity,affinity, and capacity. In some instances, Fv framework region (FR)residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, “humanized antibodies” as used hereinmay also comprise residues which are found neither in the recipientantibody nor the donor antibody. These modifications are made to furtherrefine and optimize antibody performance. The humanized antibodyoptimally also will comprise at least a portion of an immunoglobulinconstant region (Fc), typically that of a human immunoglobulin. Forfurther details, see Jones et al., Nature, 321:522-525 (1986); Reichmannet al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.,2:593-596 (1992).

Antibody “effector functions” refer to those biological activitiesattributable to the Fc region (a native sequence Fc region or amino acidsequence variant Fc region) of an antibody, and vary with the antibodyisotype. Examples of antibody effector functions include: Clq bindingand complement dependent cytotoxicity; Fc receptor binding;antibody—dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and Bcell activation. “Antibody-dependent cell-mediated cytotoxicity” or ADCCrefers to a form of cytotoxicity in which secreted Ig bound onto Fcreceptors (FcRs) present on certain cytotoxic cells (e.g., naturalkiller (NK) cells, neutrophils and macrophages) enable these cytotoxiceffector cells to bind specifically to an antigen-bearing target celland subsequently kill the target cell with cytotoxins. The antibodies“arm” the cytotoxic cells and are required for killing of the targetcell by this mechanism. The primary cells for mediating ADCC, NK cells,express FcγRIII only, whereas monocytes express FcγR1, FcγRII andFcγRIII. Fc expression on hematopoietic cells is summarized in Table 3on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9: 457-92 (1991).To assess ADCC activity of a molecule of interest, an in vitro ACDDassay, such as that described in U.S. Pat. Nos. 5,500,362 or 5,821,337may be performed. Useful effector cells for such assays includeperipheral blood mononuclear cells (PBMC) and natural killer (NK) cells.Alternatively, or additionally, ADCC activity of the molecule ofinterest may be assessed in vivo, e.g., in an animal model such as thatdisclosed in Clynes et al., PNAS USA 95:652-656 (1998).

“Fc receptor” or “FcR” describes a receptor that binds to the Fc regionof an antibody. The preferred FcR is a native sequence human FcR.Moreover, a preferred FcR is one which binds an IgG antibody (a gammareceptor) and includes receptors of the FcγRI, FcγRII, and FcγRIIIsubclasses, including allelic variants and alternatively spliced formsof these receptors, FcγRII receptors include FcγRIIA (an “activatingreceptor”) and FcγRIIB (an “inhibiting receptor”), which have similaramino acid sequences that differ primarily in the cytoplasmic domainsthereof. Activating receptor FcγRIIA contains an immunoreceptortyrosine-based activation motif (ITAM) in its cytoplasmic domain.Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-basedinhibition motif (ITIM) in its cytoplasmic domain. (see M. Daëron, Annu.Rev. Immunol. 15:203-234 (1997). FcRs are reviewed in Ravetch and Kinet,Annu. Rev. Immunol. 9: 457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126: 330-41 (1995).Other FcRs, including those to be identified in the future, areencompassed by the term “FcR” herein. The term also includes theneonatal receptor, FcRn, which is responsible for the transfer ofmaternal IgGs to the fetus. Guyer et al., J. Immunol. 117: 587 (1976)and Kim et al., J. Immunol. 24: 249 (1994).

“Human effector cells” are leukocytes which express one or more FcRs andperform effector functions. Preferably, the cells express at leastFcγRIII and perform ADCC effector function. Examples of human leukocyteswhich mediate ADCC include peripheral blood mononuclear cells (PBMC),natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils,with PBMCs and MNK cells being preferred. The effector cells may beisolated from a native source, e.g., blood.

“Complement dependent cytotoxicity” of “CDC” refers to the lysis of atarget cell in the presence of complement. Activation of the classicalcomplement pathway is initiated by the binding of the first component ofthe complement system (Clq) to antibodies (of the appropriate subclass)which are bound to their cognate antigen. To assess complementactivation, a CDC assay, e.g., as described in Gazzano-Santoro et al.,J. Immunol. Methods 202: 163 (1996), may be performed.

A “stable” formulation is one in which the protein therein essentiallyretains its physical and chemical stability and integrity upon storage.Various analytical techniques for measuring protein stability arcavailable in the art and arc reviewed in Peptide and Protein DrugDelivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y.,York, Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10: 29-90(1993). Stability can be measured at a selected temperature for aselected time period. For rapid screening, the formulation may be keptat 40° C. for 2 weeks to 1 month, at which time stability is measured.Where the formulation is to be stored at 2-8° C., generally theformulation should be stable at 30° C. or 40° C. for at least 1 monthand/or stable at 2-8° C. for at least 2 years. Where the formulation isto be stored at 30° C., generally the formulation should be stable forat least 2 years at 30° C. and/or stable at 40° C. for at least 6months. For example, the extent of aggregation following lyophilizationand storage can be used as an indicator of protein stability. Thus, a“stable” formulation may be one wherein less than about 10% andpreferably less than about 5% of the protein are present as an aggregatein the formulation. In other embodiments, any increase in aggregateformation following lyophilization and storage of the lyophilizedformulation can be determined. For example, a “stable” lyophilizedformulation may be one wherein the increase in aggregate in thelyophilized formulation is less than about 5% and preferably less thanabout 3%, when the lyophilized formulation is stored at 2-8° C. for atleast one year. In other embodiments, stability of the proteinformulation may be measured using a biological activity assay.

A “reconstituted” formulation is one which has been prepared bydissolving a lyophilized protein formulation in a diluent such that theprotein is dispersed in the reconstituted formulation. The reconstitutedformulation is suitable for administration (e.g. parenteraladministration) to a patient to be treated with the protein of interestand, in certain embodiments of the invention, may be one which issuitable for subcutaneous administration.

An “isotonic” formulation is one which has essentially the same osmoticpressure as human blood. Isotonic formulations will generally have anosmotic pressure from about 250 to 350 mOsm. The term “hypotonic”describes a formulation with an osmotic pressure below that of humanblood. Correspondingly, the term “hypertonic” is used to describe aformulation with an osmotic pressure above that of human blood.Isotonicity can be measured using a vapor pressure or ice-freezing typeosmometer, for example. The formulations of the present invention arehypertonic as a result of the addition of salt and/or buffer.

A “pharmaceutically acceptable acid” includes inorganic and organicacids which are non toxic at the concentration and manner in which theyare formulated. For example, suitable inorganic acids includehydrochloric, perchloric, hydrobromic, hydroiodic, nitric, sulfuric,sulfonic, sulfinic, sulfanilic, phosphoric, carbonic, etc. Suitableorganic acids include straight and branched-chain alkyl, aromatic,cyclic, cyloaliphatic, arylaliphatic, heterocyclic, saturated,unsaturated, mono, di- and tri-carboxylic, including for example,formic, acetic, 2-hydroxyacetic, trifluoroacetic, phenylacetic,trimethylacetic, t-butyl acetic, anthranilic, propanoic,2-hydroxypropanoic, 2-oxopropanoic, propandioic, cyclopentanepropionic,cyclopentane propionic, 3-phenylpropionic, butanoic, butandioic,benzoic, 3-(4-hydroxybenzoyl)benzoic, 2-acetoxy-benzoic, ascorbic,cinnamic, lauryl sulfuric, stearic, muconic, mandelic, succinic,cmbonic, fumaric, malic, malcic, hydroxymalcic, malonic, lactic, citric,tartaric, glycolic, glyconic, gluconic, pyruvic, glyoxalic, oxalic,mesylic, succinic, salicylic, phthalic, palmoic, palmeic, thiocyanic,methanesulphonic, ethanesulphonic, 1,2-ethanedisulfonic,2-hydroxyethanesulfonic, benzenesulphonic, 4-chorobenzenesulfonic,2-napthalene-2-sulphonic, p-toluenesulphonic, camphorsulphonic,4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic, glucohcptonic,4,4′-methylenebis-3-(hydroxy-2-ene-1-carboxylic acid), hydroxynapthoic.

“Pharmaceutically-acceptable bases” include inorganic and organic baseswere are non-toxic at the concentration and manner in which they areformulated. For example, suitable bases include those formed frominorganic base forming metals such as lithium, sodium, potassium,magnesium, calcium, ammonium, iron, zinc, copper, manganese, aluminum,N-methylglucamine, morpholine, piperidine and organic nontoxic basesincluding, primary, secondary and tertiary amine, substituted amines,cyclic amines and basic ion exchange resins, [e.g.,N(R′)₄ ⁺ (where R′ isindependently H or C₁₋₄ alkyl, e.g., ammonium, Tris)], for example,isopropylamine, trimethylamine, diethylamine, triethylamine,tripropylamine, ethanolamine, 2-diethylaminoethanol, trimethamine,dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine,hydrabamine, choline, betaine, ethylenediamine, glucosamine,methylglucamine, theobromine, purines, piperazine, piperidine,N-ethylpiperidine, polyamine resins and the like. Particularly preferredorganic non-toxic bases are isopropylamine, diethylamine, ethanolamine,trimethamine, dicyclohexylamine, choline, and caffeine.

Additional pharmaceutically acceptable acids and bases useable with thepresent invention include those which are derived from the amino acids,for example, histidino, glycine, phenylalanine, aspartic acid, glutamicacid, lysine and asparagine.

“Pharmaceutically acceptable” buffers and salts include those derivedfrom both acid and base addition salts of the above indicated acids andbases. Specific buffers and or salts include histidine, succinate andacetate.

A “lyoprotectant” is a molecule which, when combined with a protein ofinterest, significantly prevents or reduces chemical and/or physicalinstability of the protein upon lyophilization and subsequent storage.Exemplary lyoprotectants include sugars and their corresponding sugaralchohols; an amino acid such as monosodium glutamate or histidine; amethylamine such as betaine; a lyotropic salt such as magnesium sulfate;a polyol such as trihydric or higher molecular weight sugar alcohols,e.g. glycerin, dextran, erythritol, glycerol, arabitol, xylitol,sorbitol, and mannitol; propylene glycol; polyethylene glycol;Pluronics®; and combinations thereof. Additional exemplarylyoprotectants include glycerin and gelatin, and the sugars mellibiose,melezitose, raffinose, mannotriose and stachyose. Examples of reducingsugars include glucose, maltose, lactose, maltulose, iso-maltulose andlactulose. Examples of non-reducing sugars include non-reducingglycosides of polyhydroxy compounds selected from sugar alcohols andother straight chain polyalcohols. Preferred sugar alcohols aremonoglycosides, especially those compounds obtained by reduction ofdisaccharides such as lactose, maltose, lactulose and maltulose. Theglycosidic side group can be either glucosidic or galactosidic.Additional examples of sugar alcohols are glucitol, maltitol, lactitoland iso-maltulose. The preferred lyoprotectant are the non-reducingsugars trehalose or sucrose.

In preparing the reduced viscosity formulations of the invention, careshould be taken using the above enumerated excipients as well as otheradditives, especially when added at high concentration, so as to notincrease the viscosity of the formulation.

The lyoprotectant is added to the pre-lyophilized formulation in a“lyoprotecting amount” which means that, following lyophilization of theprotein in the presence of the lyoprotecting amount of thelyoprotectant, the protein essentially retains its physical and chemicalstability and integrity upon lyophilization and storage.

The “diluent” of interest herein is one which is pharmaceuticallyacceptable (safe and non-toxic for administration to a human) and isuseful for the preparation of a liquid formulation, such as aformulation reconstituted after lyophilization. Exemplary diluentsinclude sterile water, bacteriostatic water for injection (BWFI), a pHbuffered solution (e.g. phosphate-buffered saline), sterile salinesolution, Ringer's solution or dextrose solution. In an alternativeembodiment, diluents can include aqueous solutions of salts and/orbuffers.

A “preservative” is a compound which can be added to the formulationsherein to reduce bacterial action. The addition of a preservative may,for example, facilitate the production of a multi-use (multiple-dose)formulation. Examples of potential preservatives includeoctadecyldimethylbenzyl ammonium chloride, hexamethonium chloride,benzalkonium chloride (a mixture of alkylbenzyldimethylammoniumchlorides in which the alkyl groups are long-chain compounds), andbenzethonium chloride. Other types of preservatives include aromaticalcohols such as phenol, butyl and benzyl alcohol, alkyl parabens suchas methyl or propyl paraben, catechol, resorcinol, cyclohexanol,3-pentanol, and m-cresol. The most preferred preservative herein isbenzyl alcohol.

A “bulking agent” is a compound which adds mass to a lyophilized mixtureand contributes to the physical structure of the lyophilized cake (e.g.facilitates the production of an essentially uniform lyophilized cakewhich maintains an open pore structure). Exemplary bulking agentsinclude mannitol, glycine, polyethylene glycol and sorbitol. The liquidformulations of the present invention obtained by reconstitution of alyophilized formulation may contain such bulking agents. “Treatment”refers to both therapeutic treatment and prophylactic or preventativemeasures. Those in need of treatment include those already with thedisorder as well as those in which the disorder is to be prevented.

“Mammal” for purposes of treatment refers to any animal classified as amammal, including humans, domestic and farm animals, and zoo, sports, orpet animals, such as dogs, horses, rabbits, cattle, pigs, hamsters,mice, cats, etc. Preferably, the mammal is human.

A “disorder” is any condition that would benefit from treatment with theprotein. This includes chronic and acute disorders or diseases includingthose pathological conditions which predispose the mammal to thedisorder in question. Non-limiting examples of disorders to be treatedherein include carcinomas and allergies.

A “therapeutically effective amount” is at least the minimumconcentration required to effect a measurable improvement or preventionof a particular disorder. Therapeutically effective amounts of knownproteins arc well known in the art, while the effective amounts ofproteins hereinafter discovered may be determined by standard techniqueswhich are well within the skill of a skilled artisan, such as anordinary physician.

“Viscosity” as used herein may be “kinematic viscosity” or “absoluteviscosity.” “Kinematic viscosity” is a measure of the resistive flow ofa fluid under the influence of gravity. When two fluids of equal volumeare placed in identical capillary viscometers and allowed to flow bygravity, a viscous fluid takes longer than a less viscous fluid to flowthrough the capillary. If one fluid takes 200 seconds to complete itsflow and another fluid takes 400 seconds, the second fluid is twice asviscous as the first on a kinematic viscosity scale. “Absoluteviscosity”, sometimes called dynamic or simple viscosity, is the productof kinematic viscosity and fluid density:

Absolute Viscosity=Kinematic Viscosity×Density

The dimension of kinematic viscosity is L²/T where L is a length and Tis a time. Commonly, kinematic viscosity is expressed in centistokes(cSt). The SI unit of kinematic viscosity is mm²/s, which is 1 cSt.Absolute viscosity is expressed in units of centipoise (cP). The SI unitof absolute viscosity is the milliPascal-second (mPa-s), where 1 cP=1mPa-s.

II. Modes for Carrying out the Invention

A. Protein Preparation

The protein to be formulated may be produced by any known technique,such as by culturing cells transformed or transfected with a vectorcontaining nucleic acid encoding the protein, as is well known in art,or through synthetic techniques (such as recombinant techniques andpeptide synthesis or a combination of these techniques) or may beisolated from an endogenous source of the protein.

Preparation of the protein to be formulated by the method of theinvention by recombinant means may be accomplished by transfccting ortransforming suitable host cells with expression or cloning vectors andcultured in conventional nutrient media modified as appropriate forinducing promoters, selecting transformants, or amplifying the genesencoding the desired sequences. The culture conditions, such as media,temperature, pH and the like, can be selected by the skilled artisanwithout undue experimentation. In general, principles, protocols, andpractical techniques for maximizing the productivity of cell culturescan be found in Mammalian Cell Biotechnology: A Practical Approach, M.Butler, Ed. (IRL Press, 1991) and Sambrook et al., Molecular Cloning: ALaboratory Manual, New York: Cold Spring Harbor Press. Methods oftransfection are known to the ordinarily skilled artisan, and includefor example, CaPO₄ and CaCl₂ transfection, electroporation,microinjection, etc. Suitable techniques are also described in Sambrooket al., supra. Additional transfection techniques are described in Shawet al., Gene 23: 315 (1983); WO 89/05859; Graham et al., Virology 52:456-457 (1978) and U.S. Pat. No. 4,399,216.

The nucleic acid encoding the desired protein for formulation accordingto the present method may be inserted into a replicable vector forcloning or expression. Suitable vectors are publicly available and maytake the form of a plasmid, cosmid, viral particle or phage. Theappropriate nucleic acid sequence may be inserted into the vector by avariety of procedures. In general, DNA is inserted into an appropriaterestriction endonuclease site(s) using techniques known in the art.Vector components generally include, but are not limited to, one or moreof a signal sequence, an origin of replication, one or more markergenes, and enhancer element, a promoter, and a transcription terminationsequence. Construction of suitable vectors containing one or more ofthese components employs standard ligation techniques which are known tothe skilled artisan.

Forms of the protein to be formulated may be recovered from culturemedium or from host cell lysates. If membrane-bound, it can be releasedfrom the membrane using a suitable detergent or through enzymaticcleavage. Cells employed for expression can also be disrupted by variousphysical or chemical means, such as freeze-thaw cycling, sonication,mechanical disruption or cell lysing agents.

Purification of the protein to be formulated may be effected by anysuitable technique known in the art, such as for example, fractionationon an ion-exchange column, ethanol precipitation, reverse phase HPLC,chromatography on silica or cation-exchange resin (e.g., DEAE),chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, gelfiltration using protein A Sepharose columns (e.g., Sephadex® G-75) toremove contaminants such as IgG, and metal chelating columns to bindepitope-tagged forms.

B. Antibody Preparation

In certain embodiments of the invention, the protein of choice is anantibody. Techniques for the production of antibodies, includingpolyclonal, monoclonal, humanized, bispecific and heteroconjugateantibodies follow.

(i) Polyclonal Antibodies.

Polyclonal antibodies are generally raised in animals by multiplesubcutaneous (sc) or intraperitoneal (ip) injections of the relevantantigen and an adjuvant. It may be useful to conjugate the relevantantigen to a protein that is immunogenic in the species to be immunized,e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, orsoybean trypsin inhibitor. Examples of adjuvants which may be employedinclude Freund's complete adjuvant and MPL-TDM adjuvant (monophosphorylLipid A, synthetic trehalose dicorynomycolate). The immunizationprotocol may be selected by one skilled in the art without undueexperimentation.

One month later the animals are boosted with 1/5 to 1/10 the originalamount of peptide or conjugate in Freund's complete adjuvant bysubcutaneous injection at multiple sites. Seven to 14 days later theanimals are bled and the serum is assayed for antibody titer. Animalsare boosted until the titer plateaus. Preferably, the animal is boostedwith the conjugate of the same antigen, but conjugated to a differentprotein and/or through a different cross-linking reagent. Conjugatesalso can be made in recombinant cell culture as protein fusions. Also,aggregating agents such as alum are suitably used to enhance the immuneresponse.

(ii) Monoclonal Antibodies.

Monoclonal antibodies are obtained from a population of substantiallyhomogeneous antibodies, i.e., the individual antibodies comprising thepopulation are identical except for possible naturally occurringmutations that may be present in minor amounts. Thus, the modifier“monoclonal” indicates the character of the antibody as not being amixture of discrete antibodies.

For example, the monoclonal antibodies may be made using the hybridomamethod first described by Kohler et al., Nature, 256:495 (1975), or maybe made by recombinant DNA methods (U.S. Pat. No. 4,816,567).

In the hybridoma method, a mouse or other appropriate host animal, suchas a hamster, is immunized as hereinabove described to elicitlymphocytes that produce or are capable of producing antibodies thatwill specifically bind to the protein used for immunization.Alternatively, lymphocytes may be immunized in vitro. Lymphocytes thenarc fused with mycloma cells using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (Goding, MonoclonalAntibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986).

The immunizing agent will typically include the protein to beformulated. Generally either peripheral blood lymphocytes (“PBLs”) areused if cells of human origin are desired, or spleen cells or lymph nodecells are used if non-human mammalian sources are desired. Thelymphoctyes are then fused with an immortalized cell line using asuitable fusing agent, such as polyethylene glycol, to form a hybridomacell. Goding, Monoclonal antibodies: Principles and Practice, AcademicPress (1986), pp. 59-103. Immortalized cell lines are usuallytransformed mammalian cell, particularly myeloma cells of rodent, bovineand human origin. Usually, rat or mouse myeloma cell lines are employed.The hybridoma cells thus prepared are seeded and grown in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.For example, if the parental myeloma cells lack the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT or HPRT), the culture mediumfor the hybridomas typically will include hypoxanthine, aminopterin, andthymidinc (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Preferred myeloma cells are those that fuse efficiently, support stablehigh-level production of antibody by the selected antibody-producingcells, and are sensitive to a medium such as HAT medium. Among these,preferred myeloma cell lines are murine myeloma lines, such as thosederived from MOPC-21 and MPC-11 mouse tumors available from the SalkInstitute Cell Distribution Center, San Diego, Calif. USA, and SP-2cells available from the American Type Culture Collection, Rockville,Md. USA. Human myeloma and mouse-human heteromyeloma cell lines alsohave been described for the production of human monoclonal antibodies(Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., MonoclonalAntibody Production Techniques and Applications, pp. 51-63 (MarcelDekker, Inc., New York, 1987)).

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against the antigen.Preferably, the binding specificity of monoclonal antibodies produced byhybridoma cells is determined by immunoprecipitation or by an in vitrobinding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunoabsorbcnt assay (ELISA).

The binding affinity of the monoclonal antibody can, for example, bedetermined by the Scatchard analysis of Munson et al., Anal. Biochem.,107:220 (1980).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding, supra). Suitable culture media for this purpose include, forexample, D-MEM or RPMI-1640 medium. In addition, the hybridoma cells maybe grown in vivo as ascites tumors in an animal.

The immunizing agent will typically include the epitope protein to whichthe antibody binds. Generally, either peripheral blood lymphocytes(“PBLs”) are used if cells of human origin are desired, or spleen cellsor lymph node cells are used if non-human mammalian sources arc desired.The lymphocytes arc then fused with an immortalized cell line using asuitable fusing agent, such as polyethylene glycol, to form a hybridomacell. Goding, Monoclonal Antibodies: Principals and Practice, AcademicPress (1986), pp. 59-103.

Immortalized cell lines are usually transformed mammalian cells,particularly mycloma cells of rodent, bovine and human origin. Usually,rat or mouse myclome cell lines are employed. The hybridoma cells may becultured in a suitable culture medium that preferably contains one ormore substances that inhibit the growth or survival of the unfused,immortalized cells. For example, if the parental cells lack the enzymehypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), theculture medium for the hybridomas typically will include hypoxanthine,aminopterin and thymidine (“HAT medium”), which substances prevent thegrowth of HGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently,support stable high level expression of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. More preferred immortalized cell lines are murinc mycloma lines,which can be obtained, for instance, from the Salk Institute CellDistribution Center, San Diego, Calif. and the American Type CultureCollection, Rockville, Md. Human myeloma and mouse-human heteromyelomacell lines also have been described for the production of humanmonoclonal antibodies. Kozbor, J. Immunol., 133:3001 (1984); Brodeur etal., Monoclonal Antibody Production Techniques and Applications, MarcelDekker, Inc., New York, (1987) pp. 51-63.

The culture medium in which the hybridoma cells arc cultured can then beassayed for the presence of monoclonal antibodies directed against theprotein to be formulated. Preferably, the binding specificity ofmonoclonal antibodies produced by the hybridoma cells is determined byimmunoprecipitation or by an in vitro binding assay, such asradioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).Such techniques and assays are known in the art. The binding affinity ofthe monoclonal antibody can, for example, be determined by the Scatchardanalysis of Munson and Pollard, Anal. Biochem., 107:220 (1980).

After the desired hybridoma cells are identified, the clones may besubcloned by limiting dilution procedures and grown by standard methods.Goding, supra. Suitable culture media for this purpose include, forexample, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium.Alternatively, the hybridoma cells may be grown in vivo as ascites in amammal.

The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

DNA encoding the monoclonal antibodies is readily isolated and sequencedusing conventional procedures (e.g., by using oligonucleotide probesthat are capable of binding specifically to genes encoding the heavy andlight chains of murine antibodies). The hybridoma cells serve as apreferred source of such DNA. Once isolated, the DNA may be placed intoexpression vectors, which are then transfected into host cells such asE. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, ormyeloma cells that do not otherwise produce immunoglobulin protein, toobtain the synthesis of monoclonal antibodies in the recombinant hostcells. Review articles on recombinant expression in bacteria of DNAencoding the antibody include Skerra et al., Curr. Opinion in Immunol.,5:256-262 (1993) and Pliickthun, Immunol. Revs. 130:151-188 (1992).

In a further embodiment, antibodies can be isolated from antibody phagelibraries generated using the techniques described in McCafferty et al.,Nature, 348:552-554 (1990). Clackson et al., Nature, 352:624-628 (1991)and Marks et al., J. Mol. Biol., 222:581-597 (1991) describe theisolation of murine and human antibodies, respectively, using phagelibraries. Subsequent publications describe the production of highaffinity (nM range) human antibodies by chain shuffling (Marks et al.,Bio/Technology, 10:779-783 (1992)), as well as combinatorial infectionand in vivo recombination as a strategy for constructing very largephage libraries (Waterhouse et al., Nuc. Acids. Res., 21:2265-2266(1993)). Thus, these techniques are viable alternatives to traditionalmonoclonal antibody hybridoma techniques for isolation of monoclonalantibodies.

The DNA also may be modified, for example, by substituting the codingsequence for human heavy- and light-chain constant domains in place ofthe homologous murine sequences (U.S. Pat. No. 4,816,567; Morrison, etal., Proc. Natl Acad. Sci. USA, 81:6851 (1984)), or by covalentlyjoining to the immunoglobulin coding sequence all or part of the codingsequence for a non-immunoglobulin polypeptide.

Typically such non-immunoglobulin polypeptides are substituted for theconstant domains of an antibody, or they are substituted for thevariable domains of one antigen-combining site of an antibody to createa chimeric bivalent antibody comprising one antigen-combining sitehaving specificity for an antigen and another antigen-combining sitehaving specificity for a different antigen.

Chimeric or hybrid antibodies also may be prepared in vitro using knownmethods in synthetic protein chemistry, including those involvingcrosslinking agents. For example, immunotoxins may be constructed usinga disulfide-exchange reaction or by forming a thioether bond. Examplesof suitable reagents for this purpose include iminothiolate andmethyl-4-mercaptobutyrimidate.

(iii) Humanized and Human Antibodies.

The antibodies subject to the formulation method may further comprisehumanized or human antibodies. Humanized forms of non-human (e.g.,murine) antibodies are chimeric immunoglobulins, immunoglobulin chainsor fragments thereof (such as Fv, Fab, Fab′, F(ab′)₂ or otherantigen-binding subsequences of antibodies) which contain minimalsequence derived from non-human immunoglobulin. Humanized antibodiesinclude human immunoglobulins (recipient antibody) in which residuesfrom a complementarity determining region (CDR) of the recipient arereplaced by residues from a CDR of a non-human species (donor antibody)such as mouse, rat or rabbit having the desired specificity, affinityand capacity. In some instances, Fv framework residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domain,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. Jones et al., Nature 321: 522-525 (1986); Riechmann etal., Nature 332: 323-329 (1988) and Presta, Curr. Opin. Struct. Biol. 2:593-596 (1992).

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the method of Winter and co-workers,Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature332:323-327 (1988); Verhoeyen et al., Science 239:1534-1536 (1988), orthrough substituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is very important to reduceantigenicity. According to the so-called “best-fit” method, the sequenceof the variable domain of a rodent antibody is screened against theentire library of known human variable-domain sequences. The humansequence which is closest to that of the rodent is then accepted as thehuman framework (FR) for the humanized antibody. Sims et al., J.Immunol., 151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901(1987). Another method uses a particular framework derived from theconsensus sequence of all human antibodies of a particular subgroup oflight or heavy chains. The same framework may be used for severaldifferent humanized antibodies. Carter et al., Proc. Natl. Acad. Sci.USA, 89:4285 (1992); Presta et al., J. Immnol., 151:2623 (1993).

It is further important that antibodies be humanized with retention ofhigh affinity for the antigen and other favorable biological properties.To achieve this goal, according to a preferred method, humanizedantibodies arc prepared by a process of analysis of the parentalsequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the recipient and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the CDR residues aredirectly and most substantially involved in influencing antigen binding.

Alternatively, it is now possible to produce transgenic animals (e.g.,mice) that are capable, upon immunization, of producing a fullrepertoire of human antibodies in the absence of endogenousimmunoglobulin production. For example, it has been described that thehomozygous deletion of the antibody heavy-chain joining region (J_(H))gene in chimeric and germ-line mutant mice results in completeinhibition of endogenous antibody production. Transfer of the humangerm-line immunoglobulin gene array in such germ-line mutant mice willresult in the production of human antibodies upon antigen challenge.See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551(1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggermann etal., Year in Immuno., 7:33 (1993). Human antibodies can also be derivedfrom phage-display libraries (Hoogenboom et al., J. Mol. Biol., 227:381(1991); Marks et al., J. Mol. Biol., 222:581-597 (1991)).

Human antibodies can also be produced using various techniques known inthe art, including phage display libraries. Hoogenboom and Winter, J.Mol. Biol. 227: 381 (1991); Marks et al., J. Mol. Biol. 222: 581 (1991).The techniques of Cole et al., and Boerner et al., are also availablefor the preparation of human monoclonal antibodies (Cole et al.,Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) andBoerner et al., J. Immunol. 147(1): 86-95 (1991). Similarly, humanantibodies can be made by introducing human immunoglobulin loci intotransgenic animals, e.g., mice in which the endogenous immunoglobulingenes have been partially or completely inactivated. Upon challenge,human antibody production is observed, which closely resemble that seenin human in all respects, including gene rearrangement, assembly andantibody repertoire. This approach is described, for example, in U.S.Pat. Nos. 5,545,807; 5,545,806, 5,569,825, 5,625,126, 5,633,425,5,661,016 and in the following scientific publications: Marks et al.,Bio/Technology 10: 779-783 (1992); Lonberg et al., Nature 368: 856-859(1994); Morrison, Nature 368: 812-13 (1994), Fishwild et al., NatureBiotechnology 14: 845-51 (1996), Neuberger, Nature Biotechnology 14: 826(1996) and Lonberg and Huszar, Intern. Rev. Immunol. 13: 65-93 (1995).

(iv) Antibody Dependent Enzyme-Mediated Prodrug Therapy (ADEPT)

The antibodies of the present invention may also be used in ADEPT byconjugating the antibody to a prodrug-activating enzyme which converts aprodrug (e.g. a peptidyl chemotherapeutic agent, see WO 81/01145) to anactive anti-cancer drug. See, for example, WO 88/07378 and U. S. Pat.No. 4,975,278.

The enzyme component of the immunoconjugate useful for ADEPT includesany enzyme capable of acting on a prodrug in such as way so as toconvert it into its more active, cytotoxic form.

Enzymes that are useful in the method of this invention include, but arenot limited to, glycosidase, glucose oxidase, human lysozyme, humanglucuronidase, alkaline phosphatase useful for convertingphosphate-containing prodrugs into free drugs; arylsulfatase useful forconverting sulfate-containing prodrugs into free drugs; cytosinedeaminase useful for converting non-toxic 5-fluorocytosine into theanti-cancer drug 5-fluorouracil; proteases, such as serratia protease,thermolysin, subtilisin, carboxypeptidases (e.g., carboxypeptidase G2and carboxypeptidase A) and cathepsins (such as cathepsins B and L),that are useful for converting peptide-containing prodrugs into freedrugs; D-alanylcarboxypeptidases, useful for converting prodrugs thatcontain D-amino acid substituents; carbohydrate-cleaving enzymes such asβ-galactosidase and neuraminidase useful for converting glycosylatedprodrugs into free drugs; β-lactamase useful for converting drugsderivatized with β-lactams into free drugs; and penicillin amidases,such as penicillin Vamidase or penicillin G amidase, useful forconverting drugs derivatized at their amine nitrogens with phenoxyacetylor phenylacetyl groups, respectively, into free drugs. Alternatively,antibodies with enzymatic activity, also known in the art as “abzymes”can be used to convert the prodrugs of the invention into free activedrugs (see, e.g., Massey, Nature 328: 457-458 (1987)). Antibody-abzymeconjugates can be prepared as described herein for delivery of theabzyme to a tumor cell population.

The enzymes of this invention can be covalently bound to the anti-IL-17or anti-LIF antibodies by techniques well known in the art such as theuse of the heterobifunctional cross-linking agents discussed above.Alternatively, fusion proteins comprising at least the antigen bindingregion of the antibody of the invention linked to at least afunctionally active portion of an enzyme of the invention can beconstructed using recombinant DNA techniques well known in the art (see,e.g. Neuberger et al., Nature 312: 604-608 (1984)).

(iv) Bispecific and Polyspecific Antibodies

Bispecific antibodies (BsAbs) are antibodies that have bindingspecificities for at least two different epitopes. Such antibodies canbe derived from full length antibodies or antibody fragments (e.g.F(ab′)₂ bispecific antibodies).

Methods for making bispecific antibodies are known in the art.Traditional production of full length bispecific antibodies is based onthe coexpression of two immunoglobulin heavy chain-light chain pairs,where the two chains have different specificities. Millstein et al.,Nature, 305:537-539 (1983). Because of the random assortment ofimmunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of 10 different antibody molecules, of whichonly one has the correct bispecific structure. Purification of thecorrect molecule, which is usually done by affinity chromatographysteps, is rather cumbersome, and the product yields are low. Similarprocedures are disclosed in WO 93/08829 and in Traunecker et al., EMBOJ., 10:3655-3659 (1991).

Antibody variable domains with the desired binding specificities(antibody-antigen combining sites) can be fused to immunoglobulinconstant domain sequences. The fusion preferably is with animmunoglobulin heavy-chain constant domain, comprising at least part ofthe hinge, CH2, and CH3 regions. It is preferred to have the firstheavy-chain constant region (CH1) containing the site necessary forlight-chain binding present in at least one of the fusions. DNAsencoding the immunoglobulin heavy-chain fusions, and, if desired, theimmunoglobulin light chain, are inserted into separate expressionvectors, and are co-transfected into a suitable host organism. Forfurther details of generating bispecific antibodies, see, for example,Suresh et al., Methods in Enzymology 121: 210 (1986).

According to a different approach, antibody variable domains with thedesired binding specificities (antibody-antigen combining sites) arefused to immunoglobulin constant domain sequences. The fusion preferablyis with an immunoglobulin heavy chain constant domain, comprising atleast part of the hinge, CH2, and CH3 regions. It is preferred to havethe first heavy-chain constant region (CH1) containing the sitenecessary for light chain binding, present in at least one of thefusions. DNAs encoding the immunoglobulin heavy chain fusions and, ifdesired, the immunoglobulin light chain, are inserted into separateexpression vectors, and are co-transfected into a suitable hostorganism. This provides for great flexibility in adjusting the mutualproportions of the three polypeptide fragments in embodiments whenunequal ratios of the three polypeptide chains used in the constructionprovide the optimum yields. It is, however, possible to insert thecoding sequences for two or all three polypeptide chains in oneexpression vector when the expression of at least two polypeptide chainsin equal ratios results in high yields or when the ratios are of noparticular significance.

According to another approach described in WO 96/27011, the interfacebetween a pair of antibody molecules can be engineered to maximize thepercentage of heterodimers which are recovered from recombinant cellculture. The preferred interface comprises at least a part of the CH3region of an antibody constant domain. In this method, one or more smallamino acid side chains from the interface of the first antibody moleculeare replaced with larger side chains (e.g., tyrosine or tryptophan).Compensatory “cavities” of identical or similar size to the large sidechains(s) are created on the interface of the second antibody moleculeby replacing large amino acid side chains with smaller ones (e.g.,alanine or threonine) This provides a mechanism for increasing the yieldof the heterodimer over other unwanted end-products such as homodimers.

In a preferred embodiment of this approach, the bispecific antibodiesare composed of a hybrid immunoglobulin heavy chain with a first bindingspecificity in one arm, and a hybrid immunoglobulin heavy chain-lightchain pair (providing a second binding specificity) in the other arm. Itwas found that this asymmetric structure facilitates the separation ofthe desired bispecific compound from unwanted immunoglobulin chaincombinations, as the presence of an immunoglobulin light chain in onlyone half of the bispecific molecule provides for a facile way ofseparation. This approach is disclosed in WO 94/04690, published Mar 3,1994. For further details of generating bispecific antibodies see, forexample, Suresh et al., Methods in Enzymology, 121:210 (1986).

Bispecific antibodies include cross-linked or “heteroconjugate”antibodies. For example, one of the antibodies in the heteroconjugatecan be coupled to avidin, the other to biotin. Such antibodies have, forexample, been proposed to target immune system cells to unwanted cells(US Pat. No. 4,676,980), and for treatment of HIV infection (WO91/00360, WO 92/200373). Heteroconjugate antibodies may be made usingany convenient cross-linking methods. Suitable cross-linking agents arewell known in the art, and are disclosed in US Patent No. 4,676,980,along with a number of cross-linking techniques.

Techniques for generating bispecific antibodies from antibody fragmentshave also been described in the literature. The following techniques canalso be used for the production of bivalent antibody fragments which arenot necessarily bispecific. For example, Fab' fragments recovered fromE. coli can be chemically coupled in vitro to form bivalent antibodies.See, Shalaby et al., J. Exp. Med., 175:217-225 (1992).

Bispecific antibodies can be prepared as full length antibodies orantibody fragments (e.g. F(ab′)₂ bispecific antibodies). Techniques forgenerating bispecific antibodies from antibody fragments have beendescribed in the literature. For example, bispecific antibodies can beprepared using chemical linkage. Brennan et al., Science 229: 81 (1985)describe a procedure wherein intact antibodies are proteolyticallycleaved to generate F(ab′)₂ fragments. These fragments are reduced inthe presence of the dithiol complexing agent sodium arsenite tostabilize vicinal dithiols and prevent intermolecular disulfideformation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-TNB derivative to form the bispecificantibody. The bispecific antibodies produced can be used as agents forthe selective immobilization of enzymes.

Fab′ fragments may be directly recovered from E. coli and chemicallycoupled to form bispecific antibodies. Shalaby et al., J. Exp. Med. 175:217-225 (1992) describes the production of fully humanized bispecificantibody F(ab′)₂ molecules. Each Fab′ fragment was separately secretedfrom E. coli and subjected to directed chemical coupling in vitro toform the bispecific antibody. The bispecific antibody thus formed wasable to bind to cells overexpressing the ErbB2 receptor and normal humanT cells, as well as trigger the lytic activity of human cytotoxiclymphocytes against human breast tumor targets.

Various techniques for making and isolating bivalent antibody fragmentsdirectly from recombinant cell culture have also been described. Forexample, bivalent heterodimers have been produced using leucine zippers.Kostelny et al., J. Immunol., 148(5):1547-1553 (1992). The leucinezipper peptides from the Fos and Jun proteins were linked to the Fab′portions of two different antibodies by gene fusion. The antibodyhomodimers were reduced at the hinge region to form monomers and thenre-oxidized to form the antibody heterodimers. The “diabody” technologydescribed by Hollinger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448(1993) has provided an alternative mechanism for makingbispecific/bivalent antibody fragments. The fragments comprise aheavy-chain variable domain (V_(H)) connected to a light-chain variabledomain (V_(L)) by a linker which is too short to allow pairing betweenthe two domains on the same chain. Accordingly, the V_(H) and V_(L)domains of one fragment are forced to pair with the complementary V_(L)and V_(H) domains of another fragment, thereby forming twoantigen-binding sites. Another strategy for making bispecific/bivalentantibody fragments by the use of single-chain Fv (sFv) dimers has alsobeen reported. See Gruber et al., J. Immunol., 152:5368 (1994).

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147: 60(1991).

Exemplary bispecific antibodies may bind to two different epitopes on agiven molecule. Alternatively, an anti-protein arm may be combined withan arm which binds to a triggering molecule on a leukocyte such as aT-cell receptor molecule (e.g., CD2, CD3, CD28 or B7), or Fc receptorsfor IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16)so as to focus cellular defense mechanisms to the cell expressing theparticular protein. Bispccific antibocis may also be used to localizecytotoxic agents to cells which express a particular protein. Suchantibodies possess a protein-binding arm and an arm which binds acytotoxic agent or a radionuclide chelator, such as EOTUBE, DPTA, DOTAor TETA. Another bispecific antibody of interest binds the protein ofinterest and further binds tissue factor (TF).

(v) Heteroconjugate Antibodies

Heteroconjugate antibodies are also within the scope of the presentinvention. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. Such antibodies have, for example, been proposed totatget immune system cells to unwanted cells, U.S. Pat. No. 4,676,980,and for treatment of HIV infection. WO 91/00360, WO 92/200373 and EP03089. It is contemplated that the antibodies may be prepared in vitrousing known methods in synthetic protein chemistry, including thoseinvolving crosslinking agents. For example, immunotoxins may beconstructed using a disulfide exchange reaction or by forming athioether bond. Examples of suitable reagents for this purpose includeiminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, forexample, in U.S. Pat. No. 4,676,980.

B. Preparation of Lyophilized Formulations

Although the formulations herein are not limited to reconstitutedlyophilized formulations, in a particular embodiment, the proteins arelyophilized and then reconstituted to produce the reduced-viscositystable liquid formulations of the invention. In this particularembodiment, after preparation of the protein of interest as describedabove, a “pre-lyophilized formulation” is produced. The amount ofprotein present in the pre-lyophilized formulation is determined takinginto account the desired dose volumes, mode(s) of administration etc.For example, the starting concentration of an intact antibody can befrom about 2 mg/ml to about 50 mg/ml, preferably from about 5 mg/ml toabout 40 mg/ml and most preferably from about 20-30 mg/ml.

The protein to be formulated is generally present in solution. Forexample, in the elevated ionic strength reduced viscosity formulationsof the invention, the protein may be present in a pH-buffered solutionat a pH from about 4-8, and preferably from about 5-7. The bufferconcentration can be from about 1 mM to about 20 mM, alternatively fromabout 3 mM to about 15 mM, depending, for example, on the buffer and thedesired tonicity of the formulation (e.g. of the reconstitutedformulation). Exemplary buffers and/or salts are those which arepharmaceutically acceptable and may be created from suitable acids,bases and salts thereof, such as those which are defined under“pharmaceutically acceptable” acids, bases or buffers.

In one embodiment, a lyoprotectant is added to the pre-lyophilizedformulation. The amount of lyoprotectant in the pre-lyophilizedformulation is generally such that, upon reconstitution, the resultingformulation will be isotonic. However, hypertonic reconstitutedformulations may also be suitable. In addition, the amount oflyoprotectant must not be too low such that an unacceptable amount ofdegradation/aggregation of the protein occurs upon lyophilization.However, exemplary lyoprotectant concentrations in the pre-lyophilizedformulation are from about 10 mM to about 400 mM, alternatively fromabout 30 mM to about 300 mM, alternatively from about 50 mM to about 100mM. Exemplery lyoprotectants include sugars and sugar alcohols such assucrose, mannose, trehalose, glucose, sorbitol, mannitol. However, underparticular circumstances, certain lyoprotectants may also contribute toan increase in viscosity of the formulation. As such, care should betaken so as to select particular lyoprotectants which minimize orneutralize this effect. Additional lyoprotectants are described aboveunder the definition of “lyoprotectants”.

The ratio of protein to lyoprotectant can vary for each particularprotein or antibody and lyoprotectant combination. In the case of anantibody as the protein of choice and a sugar (e.g., sucrose ortrehalose) as the lyoprotectant for generating an isotonic reconstitutedformulation with a high protein concentration, the molar ratio oflyoprotectant to antibody may be from about 100 to about 1500 moleslyoprotectant to 1 mole antibody, and preferably from about 200 to about1000 moles of lyoprotectant to 1 mole antibody, for example from about200 to about 600 moles of lyoprotectant to 1 mole antibody.

In a preferred embodiment, it may be desirable to add a surfactant tothe pre-lyophilized formulation. Alternatively, or in addition, thesurfactant may be added to the lyophilized formulation and/or thereconstituted formulation. Exemplary surfactants include nonionicsurfactants such as polysorbates (e.g. polysorbates 20 or 80);polyoxamers (e.g. poloxamer 188); Triton; sodium octyl glycoside;lauryl-, myristyl-, linoleyl-, or stearyl-sulfobetaine; lauryl-,myristyl-, linoleyl- or stearyl-sarcosine; linoleyl-, myristyl-, orcetyl-betaine; lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-,myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine (e.g.lauroamidopropyl); myristamidopropyl-, palmidopropyl-, orisostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or disodiummethyl oleyl-taurate; and the MONAQUA™ series (Mona Industries, Inc.,Paterson, New Jersey), polyethyl glycol, polypropyl glycol, andcopolymers of ethylene and propylene glycol (e.g. Pluronics, PF68 etc).The amount of surfactant added is such that it reduces particulateformation of the reconstituted protein and minimizes the formation ofparticulates after reconstitution. For example, the surfactant may bepresent in the pre-lyophilized formulation in an amount from about0.001-0.5%, alternatively from about 0.005-0.05%.

A mixture of the lyoprotectant (such as sucrose or trehalose) and abulking agent (e.g. mannitol or glycinc) may be used in the preparationof the prc-lyophilization formulation. The bulking agent may allow forthe production of a uniform lyophilized cake without excessive pocketstherein etc. Other pharmaceutically acceptable carriers, excipients orstabilizers such as those described in Remington's PharmaceuticalSciences 16th edition, Osol, A. Ed. (1980) may be included in thepre-lyophilized formulation (and/or the lyophilized formulation and/orthe reconstituted formulation) provided that they do not adverselyaffect the desired characteristics of the formulation. Acceptablecarriers, excipients or stabilizers are nontoxic to recipients at thedosages and concentrations employed and include; additional bufferingagents; preservatives; co-solvents; antioxidants including ascorbic acidand methionine; chelating agents such as EDTA; metal complexes (e.g.Zn-protein complexes); biodegradable polymers such as polyesters; and/orsalt-forming countcrions such as sodium.

The formulation herein may also contain more than one protein asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect the otherprotein. For example, it may be desirable to provide two or moreantibodies which bind to the HER2 receptor or IgE in a singleformulation.

Such proteins are suitably present in combination in amounts that areeffective for the purpose intended.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes, prior to, or following, lyophilization and reconstitution.Alternatively, sterility of the entire mixture may be accomplished byautoclaving the ingredients, except for protein, at about 120° C. forabout 30 minutes, for example.

After the protein, optional lyoprotectant and other optional componentsare mixed together, the formulation is lyophilized. Many differentfreeze-dryers are available for this purpose such as Hull50™ (Hull, USA)or GT20™ (Leybold-Heraeus, Germany) freeze-dryers. Freeze-drying isaccomplished by freezing the formulation and subsequently subliming icefrom the frozen content at a temperature suitable for primary drying.Under this condition, the product temperature is below the eutecticpoint or the collapse temperature of the formulation. Typically, theshelf temperature for the primary drying will range from about −30 to25° C. (provided the product remains frozen during primary drying) at asuitable pressure, ranging typically from about 50 to 250 mTorr. Theformulation, size and type of the container holding the sample (e.g.,glass vial) and the volume of liquid will mainly dictate the timerequired for drying, which can range from a few hours to several days(e.g. 40-60 hrs). Optionally, a secondary drying stage may also beperformed depending upon the desired residual moisture level in theproduct. The temperature at which the secondary drying is carried outranges from about 0-40° C., depending primarily on the type and size ofcontainer and the type of protein employed. For example, the shelftemperature throughout the entire water removal phase of lyophilizationmay be from about 15-30° C. (e.g., about 20° C.). The time and pressurerequired for secondary drying will be that which produces a suitablelyophilized cake, dependent, e.g., on the temperature and otherparameters. The secondary drying time is dictated by the desiredresidual moisture level in the product and typically takes at leastabout 5 hours (e.g. 10-15 hours). The pressure may be the same as thatemployed during the primary drying step. Freeze-drying conditions can bevaried depending on the formulation and vial size.

C. Reconstitution of a Lyophilized Formulation

Prior to administration to the patient, the lyophilized formulation isreconstituted with a pharmaceutically acceptable diluent such that theprotein concentration in the reconstituted formulation is at least about50 mg/ml, for example from about 50 mg/ml to about 400 mg/ml,alternatively from about 80 mg/ml to about 300 mg/ml, alternatively fromabout 90 mg/ml to about 150 mg/ml. Such high protein concentrations inthe reconstituted formulation are considered to be particularly usefulwhere subcutaneous delivery of the reconstituted formulation isintended. However, for other routes of administration, such asintravenous administration, lower concentrations of the protein in thereconstituted formulation may be desired (for example from about 5-50mg/ml, or from about 10-40 mg/ml protein in the reconstitutedformulation). In certain embodiments, the protein concentration in thereconstituted formulation is significantly higher than that in thepre-lyophilized formulation. For example, the protein concentration inthe reconstituted formulation may be about 2-40 times, alternatively3-10 times, alternatively 3-6 times (e.g. at least three fold or atleast four fold) that of the pre-lyophilized formulation.

Reconstitution generally takes place at a temperature of about 25° C. toensure complete hydration, although other temperatures may be employedas desired. The time required for reconstitution will depend, e.g., onthe type of diluent, amount of excipient(s) and protein. Exemplarydiluents include sterile water, bacteriostatic water for injection(BWFI), a pH buffered solution (e.g. phosphate-buffered saline), sterilesaline solution, Ringer's solution or dextrose solution. The diluentoptionally contains a preservative. Exemplary preservatives have beendescribed above, with aromatic alcohols such as benzyl or phenol alcoholbeing the preferred preservatives. The amount of preservative employedis determined by assessing different preservative concentrations forcompatibility with the protein and preservative efficacy testing. Forexample, if the preservative is an aromatic alcohol (such as benzylalcohol), it can be present in an amount from about 0.1-2.0% andpreferably from about 0.5-1.5%, but most preferably about 1.0-1.2%.

Preferably, the reconstituted formulation has less than 6000 particlesper vial which are 10 vim in size.

D. Administration of the Formulation

The formulations of the present invention, including but not limited toreconstituted formulations, are administered to a mammal in need oftreatment with the protein, preferably a human, in accord with knownmethods, such as intravenous administration as a bolus or by continuousinfusion over a period of time, by intramuscular, intraperitoneal,intracerobrospinal, subcutaneous, intra-articular, intrasynovial,intrathecal, oral, topical, or inhalation routes.

In preferred embodiments, the formulations are administered to themammal by subcutaneous (i.e. beneath the skin) administration. For suchpurposes, the formulation may be injected using a syringe. However,other devices for administration of the formulation are available suchas injection devices (e.g. the Inject-ease™ and Genject™ devices);injector pens (such as the GenPen™); needleless devices (e.g.MediJector™ and BioJector™); and subcutaneous patch delivery systems.

The appropriate dosage (“therapeutically effective amount”) of theprotein will depend, for example, on the condition to be treated, theseverity and course of the condition, whether the protein isadministered for preventive or therapeutic purposes, previous therapy,the patient's clinical history and response to the protein, the type ofprotein used, and the discretion of the attending physician. The proteinis suitably administered to the patient at one time or over a series oftreatments and may be administered to the patient at any time fromdiagnosis onwards. The protein may be administered as the sole treatmentor in conjunction with other drugs or therapies useful in treating thecondition in question.

Where the protein of choice is an antibody, from about 0.1-20 mg/kg isan initial candidate dosage for administration to the patient, whether,for example, by one or more separate administrations. However, otherdosage regimens may be useful. The progress of this therapy is easilymonitored by conventional techniques.

Uses for an anti-IgE formulation (e.g., rhuMAbE-25, rhMAbE-26) includethe treatment or prophylaxis of IgE-mediated allergic diseases,parasitic infections, interstitial cystitis and asthma, for example.Depending on the disease or disorder to be treated, a therapeuticallyeffective amount (e.g. from about 1-15 mg/kg) of the anti-IgE antibodyis administered to the patient.

E. Articles of Manufacture

In another embodiment of the invention, an article of manufacture isprovided which contains the formulation and preferably providesinstructions for its use. The article of manufacture comprises acontainer. Suitable containers include, for example, bottles, vials(e.g. dual chamber vials), syringes (such as dual chamber syringes) andtest tubes. The container may be formed from a variety of materials suchas glass or plastic. The container holds the lyophilized formulation andthe label on, or associated with, the container may indicate directionsfor reconstitution and/or use. For example, the label may indicate thatthe lyophilized formulation is reconstituted to protein concentrationsas described above. The label may further indicate that the formulationis useful or intended for subcutaneous administration. The containerholding the formulation may be a multi-use vial, which allows for repeatadministrations (e.g. from 2-6 administrations) of the reconstitutedformulation. The article of manufacture may further comprise a secondcontainer comprising a suitable diluent (e.g. BWFI). Upon mixing of thediluent and the lyophilized formulation, the final protein concentrationin the reconstituted formulation will generally be at least 50 mg/ml.The article of manufacture may further include other materials desirablefrom a commercial and user standpoint, including other buffers,diluents, filters, needles, syringes, and package inserts withinstructions for use.

The invention will be more fully understood by reference to thefollowing examples. They should not, however, be construed as limitingthe scope of the invention. All citations throughout the disclosure archereby expressly incorporated by reference.

Example 1

The effects of protein concentrations on the viscosity of a recombinantanti-IgE monoclonal antibody formulation (rhuMAb E25) were studied at25° C. This antibody is a humanized anti-IgE monoclonal antibody thathas been developed by Genentech Inc. as a potential therapeutic agent totreat allergic rhinitis and allergic asthma (Presta et al., J. Immunol.151(5): 2623-2632 (1993)(PCT/US92/06860). The formulated rhuMAb E25 wasformulated to a final concentration of 40 mg/ml, 85 mM Sucrose, 5 mMHistidine, 0.01% Polysorbate 20 and filled into 5 cc vials. The sampleswere then frozen from 5° C. to −50° C. in 45 minutes and followed by asequencial increase in the lyophilizer shelf temperature 10° C. per hourfrom −50° C. to 25° C. A drying step was conducted at a shelftemperature of 25° C. and a chamber pressure of 50 mTorr for 39 hours.The lyophilized rhuMAb E25 was reconstituted with SWFI to produce asolution with rhuMAb E25 at 125 mg/ml, 266 mM sucrose, 16 mM histidine,0.03% polysorbate 20.

The viscosity of reconstituted samples were measured in Cannon-FenskeRoutine capillary viscometer (Industrial Research Glassware LTD). Thesamples were measured approximately at 8 ml with a glass pipette andloaded into a capillary viscometer of size 50 for liquid samples withkinematic viscosity ranging from 0.8 to 4 cs or size 200 for thoseranging from 20 to 100 cs. The sample temperature was maintained at 25°C. in a waterbath with a digitized temperature control system. Theviscometer was placed into the holder vertically and inserted into thewaterbath that was maintained at a fixed temperature. The efflux timewas measured by allowing the liquid sample to flow freely down pastmarks. The kinematic viscosity of liquid sample in centistokes wascalculated by multiplying the efflux time in seconds by the viscometerconstants (0.004 for size 50 and 0.015 for size 100). The viscosity ofE25 solution is highly dependent on the concentration of proteinmolecules (FIG. 1). It increases exponentially with increase of rhuMAbE25 concentration. At 25° C., the reconstituted rhuMAb E25 at 125 mg/mlis about 80 fold more viscous than water.

Example 2

The lyophilized rhuMAb E25 of Example 1 was reconstituted with differentconcentrations of NaCl solution. The viscosity of reconstituted solutionwas measured at 25° C. in a Cannon-Fcnskc Routine capillary viscometerusing the same method as described in Example 1.

The results as shown in FIG. 2 demonstrate that the addition of NaCl cansignificantly reduce the viscosity of the protein formulation. Thereconstituted rhuMAb E25 with 100 mM NaCl will give the solution that isabout 4 fold less viscous than that reconstituted with SWFI.

The preparation of a rhuMAb E25 lyophilized formulation with 100 mM NaClresulted in a slightly hypertonic solution. However, as reportedpreviously, strict isotonicity is not absolutely necessary in thattissue damage was detected only when at extremely high tonicity levels(1300 mOsmol/Kg).

Thus, the administration of a formulation containing higherconcentration of salt (100 mM to 200 mM NaCl resulting in osmolarity of˜600 to ˜700 mOsmol/Kg for current rhuMAb E25 lyophilized materials) ascontemplated herein, for reducing the viscosity of the formulation, doesnot appear to present a risk of tissue damage at the site ofadministration.

Example 3

The effects of different salts on the viscosity of rhuMAb E25 solutionwere studied at 25° C. The lyophilized rhuMAb E25 was firstreconstituted with SWFI to produce a solution with rhuMAb E25 at 125mg/ml, 266 mM sucrose, 16 mM histidine, 0.03% Polysorbate 20. Thesamples were then diluted with 10 mM histidine, 250 mM sucrose, pH 6.0to a final concentration of 40 mg/ml. Various concentrated salts werethen added into solution to bring the final salt concentration rangingfrom 0-200 mM. The viscosity of solution was determined in aCannon-Fenske Routine capillary viscometer using the same method asdescribed in Example 1.

The results were demonstrated in FIG. 3. Although each salt showsslightly different impact on change of viscosity, they appear to followa similar trend whereby the viscosity of the solution decreases withincrease in buffer concentration and ionic strength.

Example 4

Also studied were the effects of different buffers on viscosity ofrhuMAbE25 solution at 25° C. A liquid formulation containing 80 mg/ml ofrhuMAb E25, 50 mM histidine, 150 mM trehalose and 0.05% of Polysorbate20 was added with different amounts of histidine, acetate, or succinatebuffer components. The pH of sample was maintained at ˜6.0 for all thepreparation. The viscosity of solution was determined in a Cannon-FenskeRoutine capillary viscometer using the same method as described inExample 1.

As shown in FIG. 4, the viscosity of solution containing eitherhistidine or acetate buffers decreases with increasing bufferconcentration up to 200 mM. However, for succinate buffer, the viscosityof solution decreases only at low buffer concentration (<100 mM), butnot at high buffer concentration (>200 mM). Similar results have alsobeen observed in other buffers that contain negatively chargedmultivalent buffer components, such as phosphate, citrate and carbonate.

Example 5

This example used a second generation of anti-IgE monoclonal antibody,rhuMAb E26. This monoclonal antibody is a homologous to rhuMAb E25 withfive amino acid residue differences in CDR I region in the light chainand is described in WO 99/01556. The recombinant rhuMAb E26 was alsoexpressed in CHO cell line and purified with similar chromatographymethods as described above for rhuMAb E25. The samples were formulatedinto 5 mM histidinc and 275 mM sucrose with concentration of rhuMAb E25at 21 mg/ml. The viscosity of samples was measured at 6° C. in aCannon-Fenske Routine capillary viscometer using the same method asdescribed in Example 1.

The effects of NaCl on viscosity of rhuMAb E26 were shown in FIG. 5. Theresult demonstrates that the increase of NaCl concentration caneffectively reduce the viscosity of rhuMAb E26 solution.

Example 6

The effects of pH on viscosity of a highly concentrated anti-IgEmonoclonal antibody, rhuMAb E25 in liquid formulations have beenexamined in both hypotonic and isotonic conditions. The hypotonicsolutions were prepared by adding small amounts of 10% acetic acid or0.5 M arginine into an unbuffered rhuMAb E25 solution that has beenconcentrated to −130 mg/ml in Milli-Q water. The final concentrations oftotal buffer and salt were maintained at 17.5 mM. The hypotonicsolutions were then mixed with a small volume of 5 M NaCl to produce theisotonic solutions with final NaCl concentration around 150 mM NaCl. Theviscosity of solution was determined at 25° C. in a Cannon-FenskeRoutine capillary viscometer using the same method as described inExample 1.

As shown in FIG. 6, the viscosity of rhuMAb E25 solution is highlydependent on the pH of buffer, especially in very hypotonic solutions.The addition of ionic species, such as NaCl, can significantly reducesuch pH effects.

Example 7

The effects of pH on viscosity of a reconstituted anti-IgE monoclonalantibody, rhuMAb E25 have also been examined in the presence of otherexcipients, such astrehalose. The lyophilized rhuMAb E25 wasreconstituted with SWFI and then dialyzed against 20 mM Histidine, 250mM Trehalose, at pH 5. The protein concentration is about 94 mg/ml. ThepH of solution was adjusted with 1 M NaOH. The viscosity of solution wasdetermined at 25° C. in a Cannon-Fenske Routine capillary viscometerusing the same method as described in Example 1. The results, as shownin FIG. 7, demonstrated that the viscosity of antibody can besignificantly altered by pH of solution.

1.-55. (canceled)
 56. A stable liquid formulation comprising amonoclonal antibody in an amount of at least about 80 mg/ml and a saltand/or buffer in an amount of at least 50 mM, wherein the salt and/orbuffer is derived from arginine or histidine.
 57. The formulation ofclaim 56, comprising said salt and/or buffer in an amount of about50-200 mM.
 58. The formulation of claim 57, comprising said salt and/orbuffer in an amount of about 100-200 mM.
 59. The formulation of claim56, wherein said salt and/or buffer is in an amount of about 50 mM. 60.The formulation of claim 56, wherein said salt and/or buffer comprisesarginine.
 61. The formulation of claim 56, wherein said salt and/orbuffer is arginine hydrochloride.
 62. The formulation of claim 56,wherein said salt and/or buffer comprises histidine.
 63. The formulationof claim 56, further comprising a lyoprotectant.
 64. The formulation ofclaim 63, wherein said lyoprotectant is a sugar.
 65. The formulation ofclaim 64, wherein said sugar is sucrose or trehalose.
 66. Theformulation of claim 65, comprising said sugar in an amount of about60-300 mM.
 67. The formulation of claim 56, further comprising asurfactant.
 68. The formulation of claim 67, wherein the surfactant is apolysorbate.
 69. The formulation of claim 68, wherein the polysorbate ispolysorbate
 20. 70. The formulation of claim 56 which is a reconstitutedformulation.
 71. The formulation of claim 70, wherein the antibodyconcentration in the reconstituted formulation is about 2-40 timesgreater than the antibody concentration in the mixture beforelyophilization.
 72. The formulation of claim 56, wherein the antibody isin an amount of about 80 mg/ml to about 300 mg/ml.
 73. The formulationof claim 56, wherein the antibody is in an amount of about 90 mg/ml toabout 150 mg/ml.
 74. The formulation of claim 56 which is a liquidpharmaceutical formulation.
 75. The formulation of claim 74 which is forsubcutaneous administration.
 76. An article of manufacture comprising acontainer containing the formulation of claim ///56.
 77. The article ofmanufacture of claim 76 further comprising directions for administrationof said formulation.